The leading two causes of death listed by the World Health Organization (1998) are coronary heart disease and cerebrovascular disease. Since these diseases are largely triggered by blood clots, there is a considerable need for safe and effective thrombolytic agents (drugs capable of dissolving clots and restoring blood flow). However, blood clots also perform the essential physiological function of preventing hemorrhage by sealing injured vessels. This process is called hemostasis and most thrombolytic drugs interfere with hemostasis by inducing a hemophilia-like state. More importantly, these drugs lyse hemostatic fibrin (which seals injuries). By these two mechanism, thrombolytic therapy has carried significant hemorrhagic risk.
When intravascular blood clotting occurs an occlusive clot or thrombus forms, and blood flow is often arrested at that site. Blood clots consist largely of fibrin, which is a natural polymer that forms from fibrinogen in blood as the end-product of clotting. Depending on the location in the arterial system, i.e., heart, brain, or leg, such a clot can trigger a heart attack, stroke, or peripheral gangrene. In the venous circulation the same process can cause thrombophlebitis (deep vein thrombosis) or pulmonary embolism (lung clots). Together, these cardiovascular diseases constitute the leading causes of death and disability in industrialized countries. Since the tendency to form occlusive clots increases with age and populations are getting older, the incidence of these disorders is increasing worldwide.
Not surprisingly, blood clotting and thrombolysis have been a major focus of biomedical research over the past 30 years, and this research has produced an array of anti-clotting (anticoagulant) as well as clot-dissolving (thrombolytic) drugs. The first thrombolytic drugs to be developed were streptokinase (SK) and urokinase (UK), both of which have certain shortcomings, e.g., SK is antigenic and has limited efficacy, and both SK and UK induce non-specific effects, since they do not target blood clots, and act systemically upon constituents of healthy blood, causing the hemophilia-like state referred to above.
Tissue plasminogen activator (t-PA) was developed some years later and was one of the first biotechnology products. T-PA is non-antigenic, since it is a natural enzyme, is clot-specific (less likely to cause the hemophilia-like state), and is almost twice as effective as SK in lysing blood clots in vitro. However, when t-PA was tested clinically, it was found to induce more hemorrhagic side effects, and be associated with a higher stroke and reocclusion rate than SK, despite its superior specificity. These and other side effects caused its clinical benefits in the treatment of heart attacks to be little better than those obtained with SK.
Another thrombolytic agent, pro-urokinase (pro-UK) is a natural zymogen that activates plasminogen, to form plasmin, which in turn activates pro-UK to UK. Like t-PA, pro-UK is known to be selective for plasminogen bound to blood clots (see, e.g., Husain et al., U.S. Pat. No. 4,381,346), in contrast to UK (or SK), which activates plasminogen indiscriminately. This is a problem because of the high concentration of plasminogen in blood. Thus, pro-UK is referred to as fibrin clot-specific, or selective, whereas UK is non-specific. Pro-UK seemed better adapted to pharmacological use than SK, UK, or t-PA, because it is substantially inert in the blood (being a pro-enzyme) at physiological concentrations. Its activation is dependent on the presence of a fibrin clot or thrombus. Unfortunately, at therapeutic doses, which are significantly larger than naturally occurring concentrations, pro-UK becomes unstable and is readily converted by plasmin to UK. When this occurs, the selective mechanism of action of pro-UK is lost, and the hemophilia-like side effects and bleeding take place.
Because of such shortcomings of presently available thrombolytic agents, heart attacks are currently treated with angioplasty and stents, despite their technical complexity, cost, and associated delay in treatment of the patient. Out of the 1 to 2 million heart attacks that occur in the U.S. and Europe annually, a growing percentage is being treated by these invasive procedures. This is because a number of studies have demonstrated that the clinical outcome is better than with the commercially available thrombolytic drugs, and there is no risk of bleeding or hemorrhagic stroke.
The only new thrombolytic drugs that have appeared on the market in the past five years are mutant forms of t-PA. These mutant t-PAs have an efficacy and side effects essentially identical to those of t-PA, but can be administered as a bolus injection rather than by an extended infusion. Like t-PA and SK, these drugs are inimical to angioplasty, which, when possible, has become the treatment of choice for most heart attacks. Therefore, coronary reperfusion is delayed until a patient is brought into an adequately staffed catheterization laboratory. This takes at least 60–90 minutes, a critical time during which significant heart muscle is permanently lost due to lack of blood supply (perfusion).
Mutant forms of pro-UK are described in Liu et al., U.S. Pat. No. 5,472,692. These pro-UK mutants are said to have lower intrinsic activity than pro-UK and are more stable in plasma than native pro-UK. The pro-UK mutants are said to be used and administered as thrombolytic agents.